Systems and Methods for Culturing Algae With Bivalves

ABSTRACT

Provided herein are systems and methods for extracting lipids and/or producing biofuel from algae in marine and freshwater environments, wherein algae and bivalves are co-cultured in a system of enclosures comprising water that comprises recycled nutrients that are essential for algal growth. The system also include enclosures for culturing fishes which are used to harvest the algae.

This application is a continuation of U.S. application Ser. No.13/263,980, filed Dec. 19, 2011, which is the National Stage ofInternational Application No. PCT/US2010/031340, filed Apr. 16, 2010,which claims the benefit of U.S. Provisional Application No. 61/170,524,filed Apr. 17, 2009, each of which is incorporated by reference in itsentirety.

1. INTRODUCTION

Provided herein are systems and methods for extracting lipids and/orproducing biofuels from algae in marine and freshwater environments.

2. BACKGROUND OF THE INVENTION

The United States presently consumes about 42 billion gallons per yearof diesel for transportation. In 2007, a nascent biodiesel industryproduced 250 million gallons of a bio-derived diesel substitute producedfrom mostly soybean oil in the U.S. Biodiesel are fatty acid methylesters (FAME) made typically by the base-catalyzed transesterificationof triglycerides, such as vegetable oil and animal fats. Althoughsimilar to petroleum diesel in many physicochemical properties,biodiesel is chemically different and can be used alone (B100) or may beblended with petrodiesel at various concentrations in most modern dieselengines. However, a practical and affordable feedstock for use inbiodiesel has yet to be developed that would allow significantdisplacement of petrodiesel. For example, the price of soybean oil hasrisen significantly in response to the added demand from the biodieselindustry, thus limiting the growth of the biodiesel industry to lessthan 1% of the diesel demand.

It has been proposed to use algae as a feedstock for producing biofuel,such as biodiesel. Some algae strains can produce up to 50% of theirdried body weight in triglyceride oils. Algae do not need arable land,and can be grown with impaired water, neither of which competes withterrestrial food crops. Moreover, the oil production per acre can benearly 40 times that of a terrestrial crop, such as soybeans. Althoughthe development of algae presents a feasible option for biofuelproduction, there is a need to reduce the cost of producing the biofuelfrom algae. The fall in oil price in late 2008 places an even greaterpressure on the fledgling biofuel industry to develop inexpensive andefficient processes. Provided herein is a cost-efficient approach forgrowing algae in a large scale for production of biofuel.

3. SUMMARY OF THE INVENTION

Provided herein are methods and systems for extracting lipids and/orproducing biofuel from algae in marine and freshwater environments. Inone embodiment, provided herein are methods for culturing algaecomprising providing a source of water and a system comprisingenclosures for culturing algae and culturing bivalves; culturing algaeand bivalves in the system, whereby the level of at least one dissolvedinorganic or organic nutrient in the water in the system is increasedrelative to water from the source; and feeding the algae to the bivalvesat a rate such that the amount of algae produced in the system isgreater than when the algae is cultured in the system without thebivalves. The dissolved nutrient can be carbon dioxide, ammonia,ammonium ion, a nitrite, nitrite ion, a nitrate, nitrate ion, aphosphate, orthophosphate ion, minerals, proteins, carbohydrates, orlipids. Also provided herein are methods for producing biofuel furthercomprising harvesting the algae by feeding the algae to theplanktivorous fishes; extracting lipids from the planktivorous fishes;and polishing the lipids to form biofuel. Also provided herein aremethods for extracting lipids from planktivorous fishes for other uses,such as for human consumption.

In certain embodiments, the bivalves are cultured in a bivalvesenclosure separately from the algae cultured in a growth enclosure.Water in the bivalves enclosure that has an increased level dissolvednutrient(s) relative to the water from the source is flowed into thegrowth enclosure to sustain algae growth. Water from the growthenclosure and/or the fish enclosure are recirculated to the bivalvesenclosure. In one embodiment, the effluent from the fish enclosure isflowed to the bivalves enclosure at a rate that allows the bivalves tofeed on plankton that are not consumed by the plantivorous fishes. Theresidual plankton are generally of a size class that are not effectivelyretained or consumed by the planktivorous fish, e.g., less than 20 μm,about 10-20 μm, less than 10 μm, about 2-10 μm, less than 2 μm, andbetween 0.2-2 μm.

In another embodiment, the bivalves provided herein are used tocondition water obtained from a source prior to using the water toculture the algae in the system. The methods comprise flowing water froma source into the bivalves enclosure, prior to flowing into the growthenclosure, such that the level of dissolved inorganic nutrient(s) isincreased relative to the water from the source. The methods can furthercomprise removing zooplankton in water from the source, prior to flowingthe water from the source into the bivalves enclosure. The removal ofzooplankton can be accomplished by filtration or by allowing the waterto flow through an enclosure comprising zooplankton-feeding organisms,at a rate that allows the organisms to feed on the zooplankton presentin the water from the source. Zooplanktivorous fish, such as but notlimited to, Clupea harengus (Atlantic herring, seawater) and Catla catla(Indian major carp, freshwater) can be used.

The systems provided herein generally comprise a source of water; agrowth enclosure for culturing algae and a bivalves enclosure forculturing bivalves, wherein the growth enclosure and the bivalvesenclosure are in fluid communication; means to regulate the rate,direction, or both the rate and direction, of fluid flow between thegrowth enclosure and the bivalves enclosure. Each of the enclosures canhave a plurality of inlets and outlets to allow passage of fluids andmatters, including algae and/or fishes, between the enclosures and otherfacilities of the system. The system also comprise means for culturingthe bivalves and the algae; and means for feeding a controlled amount ofthe algae to the bivalves such that the amount of algae consumed by thebivalves do not result in a net loss of algae in the system over aperiod of time. The feeding of the bivalves can be controlled byadjusting the flow rate of an effluent from the growth enclosure to thebivalves enclosure and monitoring the concentration of the algae in theeffluent. The systems can further comprise means for harvesting thealgae by the planktivorous fishes; means for gathering the planktivorousfishes from which lipids are extracted and converted to biofuel; meansfor extracting lipids from the planktivorous fishes; and means forpolishing the lipids to form biofuel.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary schematic of water flow and trophic levelassociations in the bivalves-enhanced algae culture system providedherein, which includes bivalves that supply carbon dioxide, anddissolved inorganic and organic nutrients, and filter plankton of a sizesmaller than those grazed by planktivorous fishes.

FIG. 2 shows another exemplary schematic of a systems provided herein,wherein algae less than 10 to 20 micrometers are not efficientlyconsumed by the planktivous fishes and are flowed into the bivalvesenclosure for removal by the bivalves.

5. DETAILED DESCRIPTION OF THE INVENTION

Provided herein are systems and methods for growing algae for extractinglipids and/or production of biofuel. The methods provided hereincomprise growing algae in a system that comprises bivalves. The algae,fishes, and bivalves are cultured under conditions that promote nutrientrecycling, resulting in gains in algal biomass as well as fish andbivalves biomass. Unlike aquaculture operations where algae is grown asfeed for marine invertebrate larvae, the algae provided herein arecultured to accumulate lipids for use as energy feedstocks. The algaeare harvested by fish, and the lipids extracted from fish are used tomake biofuel. The bivalves cultured in the operation can optionally besold, as animal feed or human food depending on the species and themarket. Depending on the location of the system, the methods providedherein can also be used to remediate eutrophic zones, and enable carboncapture.

Using algae to produce energy feedstock at an industrial scale requiresa cost-effective and sustainable supply of nutrients. The scale of algaeculture required dwarfs those of existing culture systems that aredesigned to produce nutritional supplement, fine chemicals, oraquaculture feed. The costs of carbon, nitrogen and phosphorous must becarefully managed, and any savings and recycling of these nutrients canbe significant for a culture system at this scale. The culture systemmust also be efficient in yielding algal biomass, and remain stabledespite seasonal and other environmental changes. The bivalves in thealgae culture systems provide various benefits in these respects, andgenerally promote the efficient turnover of nutrients within the system.Without being bound by any theory, the bivalves that are cultured in thesystem serve as a supplementary source of carbon dioxide, nitrogenand/or phosphorous.

Carbon nutrition is a significant component in the operating cost of acommercial algal culture facility. Photosynthetic algae take updissolved CO₂ (inorganic carbon) and inorganic nutrients, and produceorganic carbon in the form of algal biomass. For high rates ofautotrophic production, atmospheric CO₂ alone cannot satisfy therequirement. The diffusion rate of CO₂ from atmosphere into open pondshave been estimated to sustain productivities at about 10 g dry weightper m² per day. In one embodiment, bivalves contribute carbon dioxide tothe system, which carbon dioxide is produced as a result of respirationand formation of bivalves shells.

In surface seawater with a pH of about 8.1, about 90% of the inorganiccarbon is bicarbonate ion, 9% is carbonate ion, and only 1% is dissolvedCO₂. The equilibrium is governed by a series of reactions:

CO₂ atm

CO₂ aq+H₂O

H₂CO₃H

H⁺+HCO₃ ⁻

2H⁺+CO₃ ²⁻

The bicarbonate-carbonate buffer system is important in freshwater andmarine cultures for controlling pH and can be used to provide CO₂ forphotosynthesis. When CO₂ is dissolved in water, it forms weak carbonicacid which then dissociate by losing hydrogen ions to form bicarbonate(HCO₃ ⁻) and carbonate (CO₃ ²) ions. Uptake of CO₂ for photosynthesisand its release by respiration are major processes by which organismsalter the concentration of CO₂. The bicarbonate-carbonate buffer systemin water can provide CO₂ for photosynthesis through the followingreactions:

2HCO₃ ⁻

CO₃ ²⁻+H₂O+CO₂

HCO₃ ⁻

OH⁻+CO₂

CO₃ ²⁻+H₂O

CO₂+2OH⁻

As a result of CO₂ fixation, hydroxyl groups are produced which lead toa gradual increase in pH in the water. Sparging CO₂ directly into thealgal culture is commonly used to control pH and provide carbonnutrition. However, for shallow suspensions at near neutral pH, theresidence time of the CO₂ bubbles are insufficient for dissolution,resulting in continuous loss to the atmosphere. Certain methods providedherein can reduce the operating cost of an algae facility by providing asupplementary source of CO₂ for the algal culture. The bivalves areparticularly beneficial when carbon dioxide is a limiting factor attimes in primary production, such as, in eutrophic water where nitrogenand phosphorous is not limiting.

Precipitation and dissolution of calcium carbonate in the shells andstructural components of bivalves plays a role in the cycling ofinorganic carbon. When calcium carbonate is precipitated, CO₂ isgenerated rather than consumed. Calcification induces shifts in theseawater carbonate equilibrium to generate dissolved CO₂ from inorganiccarbon. The deposition of one mole of calcium carbonate (Ca²⁺+2 HCO₃⁻→CaCO₃+H₂O+CO₂) releases nearly one mole of CO₂ in freshwater and 0.6mole CO₂ in seawater (Ware et al., 1991, Coral Reefs 11:1270-120). Thewater becomes more acidic due to the removal of bicarbonate andcarbonate ions. The increase in partial pressure of CO₂ from biogeniccalcification has been studied (Berger et al., 1982, Naturwissenschaften69:87-88), and it has been confirmed that calcification and respirationof invasive bivalves is a biogenic source of CO₂ (Chauvaud et al., 2003,Limnol Oceangr. 48:2086-2092).

Calcium is a necessary component for shell secretion and formation. Manyorganisms do not survive when the level of calcium in the water columnfalls below a threshold which is about 5 mg/l. Depending in part on thespecies being used, the minimum concentration of calcium required rangesfrom about 25 to 150 mg/l. Calcifying organisms, such as bivalves, exerta variable degree of control over the process of calcification. Growthof the shell of bivalves is governed by the synthesis of an organicmatrix and CaCO₃ crystal growth on the matrix. The amount of calcium inthe shell is a function of the calcium level in ambient water, theamount of water pumped across the animal's tissues, the local pH andbuffering capacity, the metabolism of the animal, and temperature thatcontrols the rate of the physiologic processes.

Nitrogen is an element that is after carbon the most important and oftenlimiting element to aquatic organisms. Compared to carbon that has oneinorganic form, nitrogen exists in several forms in the environment,i.e., ammonium, nitrite, nitrate, dissolved nitrogen gas. Nitrogen alsoexists as dissolved organic nitrogen and particulate organic nitrogen inthe system. Input of nitrogen from fixation of atmospheric nitrogen bybacteria is not sufficient to support the needs of a growing algalculture. Phosphorous is essential for the metabolic process of energytransfer in both photosynthesis and respiration. In aquaticenvironments, phosphorous is found in the water column as dissolvedinorganic phosphorous (usually orthophosphate), as dissolved organicphosphorous, and as particulate organic phosphorous. Phosphate releasefrom sediments is affected by adsorption-desorption equilibrium ofphosphate with iron hydroxides. When the sediments are anoxic, thestrongly reducing environment is conducive to release of phosphate intowater. Traditional fishery practices address the shortfall by addingfertilizers and/or manure to the water.

Bivalves filter water continuously at a high rate (up to 8 gallons perhour), and can thus recycle nutrients that are limiting in the system,thereby increasing primary production. Bivalves process nutrientmaterials by consuming pelagic or suspended particulate organic matters,and excreting dissolved organic and inorganic matters. Particulateorganic matters include phytoplankton, zooplankton, bacteria, anddetritus. Suspended organic detritus often has bacteria attached, andthese bacteria are decomposing as well as assimilating the carbon in thedetritus. Dissolved inorganic matters include but is not limited toammonia and orthophosphates. Dissolved organic matters, include but isnot limited to amino acids, proteins, carbohydrates, and lipids. In oneembodiment, dissolved organic and inorganic materials produced bybivalves provided herein and released in the water of the system arereadily taken up by the algae for growth. Exemplary species of bivalvesinclude but are not limited to Mytilus edulis (marine mussels),Crussostrea species (oysters), Luniellidens marginalis (freshwatermussels).

A number of feeding mechanisms have evolved in bivalves. Filter-feedingbivalves pump large volume of water and filter this water to removesuspended particulate materials. A variety of particles of differentqualitative nutrient values in the water are pumped through the bivalvegills acting as filters. Usually water enters the mantle cavity throughthe inhalant siphon, moves over the gills, and leaves through theexhalent siphon. Particles that are too large are rejected aspseudofeces and particles that are too small pass through the gills. Therejected particles are wrapped in mucus, and are expelled without havingpassed through the digestive tract. The pseudofeces sink and becomeavailable as nutrients for use by benthic organisms and bacteria.Bivalves, and pseudofeces-producing bivalves in particular, are veryefficient in the transfer of suspended particulate matters from thepelagic environment to the benthic environment. Deposit-feeding bivalvesgenerally inhabit muddy benthic environment, remove deposited sedimentsfrom the benthic environment to extract organic matter, and releaseinorganic wastes. In coastal water, a sizeable proportion of nutrientsfor primary production is provided by mineralization of particulatematter by benthic organisms. Depending on the environment,filter-feeding bivalves and/or deposit-feeding bivalves can be used inthe systems provided herein. Feces and pseudofeces, produced by thebivalves falls to and enrich the sediments surrounding the bivalveswhere benthic organisms, mostly bacteria, decompose the organic mattersand release inorganic nutrients which can be used by the algae in thesystem. Metabolic activities of the benthic organisms consume oxygen andsustain a hypoxic, reducing environment in the sediment which favors therelease of soluble inorganic phosphate from Fe(III)-bound phosphatecomplexes. The released dissolved phosphate can be used by the algae ofthe system.

In certain embodiments, the algae produced in the culture system areharvested by planktivorous fishes, such as but not limited to Brevoortiapatronus (Gulf menhaden), Sardinella longiceps (Indian oil sardine), andHypophalmichthys molitrix (Chinese silver carp). As prey size is a majorfactor affecting planktivore predation, the age or ontogenetic form ofthe fishes are selected such that the eyesight, locomotion, mouthdimensions, dentition, and gut dimension permit the fishes to ingestefficiently the dominant species of algae cultured in the system.However, there are smaller plankton in the water that pass through thegills of the selected plantivorous fishes, and thus remain in the water.The plankton in this size class includes phytoplankton, zooplankton andbacteria. A growing abundance of such smaller residual plankton, if leftunchecked in the water of the system, can compete with other algae fornutrients and light, and can also encourage the expansion of azooplankton population in the system. The occurrence of such events canmake the system less efficient, destabilize the system by changing thetrophic structure, and lead to a collapse of the algae culture. Theremoval of plankton in this size class from the water by the bivalvescan prevent accumulation or overgrowth of such plankton, and thusmaintain stability of the systems provided herein. In anotherembodiment, the bivalves stabilize the systems provided herein byfiltering plankton that are too small to be harvested efficiently by thefishes in the system.

Algae inhabit all types of aquatic environment, including but notlimited to freshwater, marine, and brackish environment. Accordingly, incertain embodiments, provided herein are selected species of algae andbivalves in any of such aquatic environments. The algal culture cancomprise a population of algae of one or more species, and thepopulation of bivalves can comprise a single species of bivalves ormultiple species. The term “algal composition” refers to any compositionthat comprises algae and is not limited to the culture in which thealgae are cultivated. It is contemplated that an algal composition canbe prepared by mixing different algae from a plurality of algalcultures. As used herein, the term “growth enclosure” refers to a waterenclosure in which the algae are grown. The majority of algal growthtakes place in the growth enclosure which is designed and equipped tooptimize algal growth. In addition, the systems provided herein cancomprise, independently and optionally, enclosures for culturing fishesthat harvest the algae, enclosures for culturing bivalves, enclosuresfor preparing a starter algal culture, enclosures in which zooplanktonis removed, bivalves husbandry units, and biomass storage units. Thebivalves husbandry units provide the environments in which the bivalvesare stocked, bred, incubated, or maintained.

The algae and the bivalves that are used in the methods provided hereinare described in Section 5.1 and 5.2 respectively. As used herein theterm “system” refers to the installations for practicing the methodsprovided herein. The methods and systems provided herein for culturingalgae are described in Section 5.3. Technical and scientific terms usedherein have the meanings commonly understood by one of ordinary skill inthe art, unless otherwise defined. Reference is made herein to variousmethodologies known to those of skill in the art. Publications and othermaterials setting forth such known methodologies to which reference ismade are incorporated herein by reference in their entireties as thoughset forth in full. The practice of the methods and systems providedherein will employ, unless otherwise indicated, techniques of chemistry,biology, and the aquaculture industry, which are within the skill of theart. Such techniques are explained fully in the literature, e.g.,Estuarine and Marine Bivalve Mollusk Culture, Menzel B. 1991, CRC press,Boca Raton, Fla., USA; Handbook of Microalgal Culture, edited by AmosRichmond, 2004, Blackwell Science; Limnology: Lake and River Ecosystems,Robert G. Wetzel, 2001, Academic Press, each of which are incorporatedby reference in their entireties.

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise. The term “about,” as used herein, unless otherwise indicated,refers to a value that is no more than 20% above or below the valuebeing modified by the term. For clarity of disclosure, and not by way oflimitation, the detailed description is divided into the subsectionswhich follow.

As used herein the term “algae” refers to any organisms with chlorophylland a thallus not differentiated into roots, stems and leaves, andencompasses prokaryotic and eukaryotic organisms that arephotoautotrophic or photoauxotrophic. The terms “microalgae” and“phytoplankton,” used interchangeably herein, refer to any microscopicalgae, photoautotrophic or photoauxotrophic protozoa, and cyanobacteria(formerly classified as Cyanophyceae). The use of the term “algal” alsorelates to microalgae and thus encompasses the meaning of “microalgal.”These microscopic aquatic organisms are also encompassed by the term“plankton.” While the term plankton includes both phytoplankton andzooplankton, it is contemplated that certain embodiments provided hereincan be practiced without isolation of the phytoplankton or removal ofthe zooplankton. The culturing and harvesting methods provided hereinare applicable to a body of water comprising plankton and fishes.

The algae that are cultured or harvested by the methods provided hereinare grown using solar power as its energy source, although algae canalso be grown under artificial light and be similarly harvested. Thealgae provided herein can be a naturally occurring species. The algaecan be a transgenic strain, a genetically manipulated strain, or aselected strain, that bears certain beneficial traits, such as but notlimited to, increased growth rate, lipid accumulation, favorable lipidcomposition, adaptation to certain environment, and robustness inchanging environmental conditions. In some embodiments, it is desirablethat the algae accumulate excess lipids and/or hydrocarbons. In otherembodiments, this is not a requirement since algal cells with relativelylower lipids level but higher carbohydrate level can also be useful,because the carbohydrates can be converted to lipids metabolically bythe harvesting fish.

Algae, including microalgae, inhabit all types of aquatic environment,including but not limited to freshwater (less than about 0.5 parts perthousand (ppt) salts), brackish (about 0.5 to about 31 ppt salts),marine (about 31 to about 38 ppt salts), and briny (greater than about38 ppt salts) environment. As certain embodiments provided herein can bepracticed in any of such aquatic environments, freshwater species,marine species, and/or species that thrive in varying and/orintermediate salinities or nutrient levels, can be used. The algae usedin the algal culture can be obtained initially from environmentalsamples of natural or man-made environments, and may contain a mixtureof prokaryotic and eukaryotic organisms, wherein some of the minorspecies may be unidentified. Freshwater filtrates from rivers, lakes;seawater filtrates from coastal areas, oceans; water in hot springs orthermal vents; and lake, marine, or estuarine sediments, can be used tosource the algae. The samples may also be collected from local or remotebodies of water, including surface as well as subterranean water.

Endemic or indigenous species are generally preferred over introducedspecies where an open farming system is used. Endemic or indigenousspecies may be enriched or isolated from water samples obtained locally(relative to the site of the culture system). It can also be beneficialto deploy algae and fishes from a local aquatic trophic system in theharvesting methods provided herein. Depending on the location of thealgae culture system, algae obtained from tropical, subtropical,temperate, polar or other climatic regions can be used.

According to certain embodiments provided herein, one or more species ofalgae will be present in the algal culture, or the algal compositionthat is to be harvested by fish. In one embodiment, the algal culture isa monoculture, wherein only one species of algae is grown. However, inmany open systems, it may be difficult to avoid the presence of otheralgae in the water. Accordingly, a monoculture may comprise about 0.1%to 2% of algae species other than the intended species. In anotherembodiments, the algal culture is a mixed culture that comprises one orseveral species of algae, i.e., the algal culture is not a monoculture.In certain embodiments, especially when an open system is in use, thecomposition of algae in the algal culture can change seasonally; thebody of water can comprise zooplankton. The algae in an algal cultureprovided herein may not all be cultivable under laboratory conditions.Not all the algae in an algal culture provided herein have to betaxonomically classified or characterized in order to be utilized incertain embodiments provided herein. Algal cultures and algalcompositions can generally be distinguished by the relative proportionsof the major groups of algae that are present.

Chlorophyll a is a commonly used indicator of algal biomass. However, itis subjected to variability of cellular chlorophyll content (0.1 to 9.7%of fresh algal weight) depending on algal species. An estimated biomassvalue can be calibrated based on the chlorophyll content of the dominantspecies within a population. Published correlation of chlorophyll aconcentration and biomass value can be used in certain embodimentsprovided herein. Generally, chlorophyll a concentration is to bemeasured within the euphotic zone of a body of water. The euphotic zoneis the depth at which the light intensity of the photosyntheticallyactive spectrum (400-700 nm) equals 1% of the subsurface lightintensity.

A mixed algal culture provided herein comprises one or several dominantspecies of macroalgae and/or microalgae. Microalgal species can beidentified by microscopy and enumerated by counting or flow cytometry,which are techniques well known in the art. A dominant species is onethat ranks high in the number of algal cells, e.g., the top one to fivespecies with the highest number of cells relative to other species. Theone or several dominant algae species may constitute greater than about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, about 95%, about 97%, about 98% of the algaepresent in the culture. In certain embodiments, several dominant algaespecies may each independently constitute greater than about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% orabout 90% of the algae present in the culture. Many other minor speciesof algae may also be present in such culture but they may constitute inaggregate less than about 50%, about 40%, about 30%, about 20%, about10%, or about 5% of the algae present. In various embodiments, one, two,three, four, or five dominant species of algae are present in a culture.Accordingly, a mixed algal culture or an algae composition can bedescribed and distinguished from other cultures or compositions by thedominant species of algae present. The composition and culture can befurther described by the percentages of cells that are of dominantspecies relative to minor species, or the percentages of each of thedominant species. The identification of dominant species can also belimited to species within a certain size class, e.g., from about 200 to2000 μm, about 20 to 200 μm, about 2 to 20 μm, or below 2 μm. It is tobe understood that mixed algal cultures or compositions having the samegenus or species of algae may be different by virtue of the relativeabundance of the various genus and/or species present.

It is contemplated that many different algal cultures or bodies of waterwhich comprise plankton, can be cultured and/or harvested efficiently bythe methods provided herein. Microalgae are preferably used in certainembodiments provided herein; while macroalgae can be less preferred incertain embodiments. In specific embodiments, algae of a particulartaxonomic group, genera or species, may be less preferred. Such algae,including one or more that are listed below, may be specificallyexcluded as a dominant species in a culture. However, it should also beunderstood that in certain embodiments, such algae may be present as acontaminant especially in an open system, or as a non-dominant group orminor species. Such algae may be present in negligent numbers, orsubstantially diluted given the volume of the culture. The presence ofsuch algal genus or species in a culture or a body of water comprisingplankton is distinguishable from cultures or other bodies of water wheresuch genus or species are dominant, or constitute the bulk of the algae.

In certain embodiments, an algal composition comprising a combination ofdifferent groups of algae can be used. The algal composition can beprepared by mixing a plurality of different algal cultures. Thedifferent groups of algae can be present in defined proportions. Thecombination and proportion of different algae in the algal compositioncan be designed to enhance the growth and/or accumulation of lipids ofcertain groups or species of fish.

In certain embodiments, one or more species of algae belonging to thefollowing phyla can be cultured and/or harvested by the methods providedherein: Cyanobacteria, Cyanophyta, Prochlorophyta, Rhodophyta,Glaucophyta, Chlorophyta, Dinophyta, Ctyptophyta, Chtysophyta,Plymnesiophyta (Haptophyta), Bacillariophyta, Xanthophyta,Eustigmatophyta, Rhaphidophyta, and Phaeophyta. In certain embodiments,algae in multicellular or filamentous forms, such as seaweeds ormacroalgae, many of which belong to the phyla Phaeophyta or Rhodophyta,are less preferred. In many embodiments, algae that are microscopic, arepreferred. Many such microalgae occurs in unicellular or colonial form.

In certain embodiments, the algal culture or the algal composition to beharvested by the methods provided herein comprises cyanobacteria (alsoknown as blue-green algae) from one or more of the following taxonomicgroups: Chroococcales, Nostocales, Oscillatoriales, Pseudanabaenales,Synechococcales, and Synechococcophycideae. Non-limiting examplesinclude Gleocapsa, Pseudoanabaena, Oscillatoria, Microcystis,Synechococcus and Arthrospira species.

In certain embodiments, the algal culture or the algal composition to beharvested comprises algae from one or more of the following taxonomicclasses: Euglenophyceae, Dinophyceae, and Ebriophyceae. Non-limitingexamples include Euglena species and the freshwater or marinedinoflagellates.

In certain embodiments, the algal culture or the algal composition to beharvested comprises green algae from one or more of the followingtaxonomic classes: Micromonadophyceae, Charophyceae, Ulvophyceae andChlorophyceae. Non-limiting examples include species of Borodinella,Chlorella (e.g., C. ellipsoidea), Chlamydomonas, Dunaliella (e.g., D.salina, D. bardawil), Franceia, Haematococcus, Oocystis (e.g., O. parva,O. pustilla), Scenedesmus, Stichococcus, Ankistrodesmus (e.g., A.falcatus), Chlorococcum, Monoraphidium, Nannochloris and Botryococcus(e.g., B. braunii). In certain embodiments, Chlamydomonas reinhardtiiare less preferred.

In certain embodiments, the algal culture or the algal composition to beharvested comprises golden-brown algae from one or more of the followingtaxonomic classes: Chtysophyceae and Synurophyceae. Non-limitingexamples include Boekelovia species (e.g. B. hooglandii) and Ochromonasspecies.

In certain embodiments, the algal culture or the algal composition to beharvested comprises freshwater, brackish, or marine diatoms from one ormore of the following taxonomic classes: Bacillariophyceae,Coscinodiscophyceae, and Fragilariophyceae. Preferably, the diatoms arephotoautotrophic or auxotrophic. Non-limiting examples includeAchnanthes (e.g., A. orientalis), Amphora (e.g., A. coffeifbrmisstrains, A. delicatissima), Amphiprora (e.g., A. hyaline), Amphipleura,Chaetoceros (e.g., C. muelleri, C. gracilis), Caloneis, Camphylodiscus,Cyclotella (e.g., C. cryptica, C. meneghiniana), Cricosphaera, Cymbella,Diploneis, Entomoneis, Fragilaria, Ilantschia, Gyrosigma, Melosira,Navicula (e.g., N. acceptata, N. biskanterae, N. pseudotenelloides, N.saprophila), Nitzschia (e.g., N. dissipata, N. communis, N. inconspicua,N. pusilla strains, N. microcephala, N. intermedia, N. hantzschiana, N.ulexandrina, N. quadrangula), Phaeoductylum (e.g., P. tricornutum),Pleurosigma, Pleurochtysis (e.g., P. carterae, P. dentata), Selenastrum,Surirella and Thalassiosira (e.g., T. weissflogii).

In certain embodiments, the algal culture or the algal composition to beharvested comprises planktons that are characteristically small with adiameter in the range of 1 to 10 μm, or 2 to 4 μm. Many of such algaeare members of Eustigmatophyta, such as but not limited toNannochloropsis species (e.g. N. salina).

In certain embodiments, the algal culture or the algal composition to beharvested comprises one or more algae from the following groups:Coelastrum, Chlorosarcina, Micractinium, Porphyridium, Nostoc,Closterium, Elakatothrix, Cyanosarcina, Trachelamonas, Kirchneriella,Carteria, Crytomonas, Chlamydamonas, Planktothrix, Anahaena,Hymenomonas, Isocluysis, Pavlova, Monodus, Monallanthus, Platymonas,Pyramimonas, Stephanodiscus, Chroococcus, Staurastrum, Netrium, andTetraselmis.

In certain embodiments, any of the above-mentioned genus and species ofalgae may independently be less preferred as a dominant species in, orexcluded from, an algal composition provided herein.

5.2 Bivalves

As used herein, the term bivalves refers to any exoskeleton-bearinginvertebrate animals of the class Bivalvia in phylum Mollusca. The termis not limited to species that are consumed by humans as food. Whenreferring to a plurality of organisms, the term “bivalve” is usedinterchangeably with the term “bivalves” regardless of whether one ormore than one species are present, unless clearly indicated otherwise.In certain embodiments, bivalves useful for the methods and systemsprovided herein can be obtained from hatcheries or collected from thewild. The bivalves may be spats, larvae, trochophores, veligers,pediveligers, juveniles, or mature bivalves. The bivalves may reproducein an enclosure within the system but not necessarily in the sameenclosure as the algae culture. Any bivalves aquaculture techniquesknown in the art can be used to stock, maintain, reproduce, and harvestthe bivalves useful for the methods and systems provided herein.

According to certain embodiments provided herein, the algae reside inthe same body of water as a population of bivalves. In one embodiment,the bivalves population comprises only one species of bivalves. Inanother embodiment, the bivalves population is mixed and thus comprisesone or several major species of bivalves. A major species is one thatranks high in the head count, e.g., the top one to five species with thehighest head count relative to other species. The one or several majorbivalves species may constitute greater than about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%,about 90%, about 95%, about 97%, about 98% of the bivalves present inthe population. In certain embodiments, several major bivalves speciesmay each constitute greater than about 10%, about 20%, about 30%, about40%, about 50%, about 60%, about 70%, or about 80% of the bivalvespresent in the population. In various embodiments, one, two, three,four, five major species of bivalves are present in a population.Accordingly, a mixed bivalves population or culture can be described anddistinguished from other populations or cultures by the major species ofbivalves present. The population or culture can be further described bythe percentages of the major and minor species, or the percentages ofeach of the major species. It is to be understood that mixed cultureshaving the same genus or species may be different by virtue of therelative abundance of the various genus and/or species present.

Bivalves inhabits most types of aquatic environment, including but notlimited to freshwater, marine, brackish, and briny environment. Incertain embodiments, as the methods and systems provided herein can bepracticed in any of such aquatic environments, any freshwater species,stenohaline species, euryhaline species, marine species, species thatgrow in brine, and/or species that thrive in varying and/or intermediatesalinities, can be used. Bivalves from tropical, subtropical, temperate,polar, and/or other climatic regions can be used. Bivalves that livewithin the following temperature ranges can be used: below 10° C., 9° C.to 18° C., 15° C. to 25° C., 20° C. to 32° C. In one embodiment,bivalves indigenous to the region at which the methods provided hereinare practiced, are used. Preferably, bivalves from the same climaticregion, same salinity environment, or same ecosystem, as the algae areused.

Bivalves use several mechanisms for feeding. Some species can use morethan one mechanism to feed, depending on the conditions of theenvironment. In one embodiment, the population of bivalves comprisesexclusively or predominantly filter feeding species. In anotherembodiment, the population of bivalves comprises exclusively orpredominantly deposit feeding species. In yet another embodiment, thepopulation of bivalves comprises exclusively or predominantly speciesthat contains photoautotrophic symbionts (zooxanthellae) and/orchemotrophic symbionts. The chemotrophic symbionts are mostly bacteriathat reside in extracellular or intracellular locations. Thechemotrophic symbionts include lithotrophic and methanotrophicorganisms. In a preferred embodiment, the major species of bivalves inthe population are not phytoplanktivores that would consume significantamounts of the growing algae. In certain mixed bivalves populationprovided herein, filter feeding and deposit feeding species are bothpresent. In addition to filter feeding and deposit feeding species,species that contain photoautotrophic and/or chemoautotrophic symbiontsare also present in a mixed bivalves population provided herein.

Bivalves from different taxonomic groups can be used in the enclosure.It should be understood that, in various embodiments, bivalves within ataxonomic group, such as a family or a genus, can be usedinterchangeably in various methods provided herein. Certain embodimentsprovided herein are described below using common names of bivalvesgroups and bivalves, as well as the scientific names of exemplaryspecies. Databases, such as the Ocean Biogeographic Information Systemby World Wide Web electronic publication, www.iobis.org, version (March2009), provide additional useful bivalves species within each of thetaxonomic groups that are useful in certain embodiments provided herein.It is contemplated that one of ordinary skill in art could, consistentwith the scope of certain embodiments provided herein, use the databasesto specify other species within each of the described taxonomic groupsfor use in the methods provided herein.

The selection of bivalves for use in the methods provided herein dependson a number of factors. Some of the most important factors are thecompatibility of the cultured algae and the bivalves. Preferably, thealgae culture grows well using the carbon dioxide and metabolic wasteproduced by the selected bivalves, thereby reducing the need to add CO₂and to fertilize the water. Preferably, the population of bivalves isself-sustaining and does not require extensive husbandry efforts topromote reproduction and rear the larvae. The methods provided hereincan employ species of bivalves that are used as human food, to offsetthe cost of operating the algae culture.

A representative group of bivalves that are useful in certainembodiments provided herein is the mollusks. Mollusks in the classes,Bivalvia and Gastropoda, are of particular interest. Within the class ofbivalves are several orders which include many ecologically andcommercially important species, such as but not limited to, Mytiloida(Mytilidae (sea mussels)); Ostreoida (Pectimidae, Ostreidae, Unionoida(freshwater mussels); Myoida, Pterioida (Pteriidae, pearl oysters), andVeneroida (Tridacnidae, Lucinidae). Within Gastropoda are subclasses oforganisms that produce pearl, e.g., Patellogastropoda, Vetigastropoda,Littprinoida, Tonnida, Muricoida and Pulmonata.

The bivalves used in the methods provided herein are preferably sessileor sedentary and of commercial value, including but not limited tooysters, mussels, scallops, and clams. Exemplary species include but arenot limited to, Crassostrea species such as C. gigas, C. virginica, C.ariakensis, C. rivularis, C. angulata, C. eradelie, C. commercialis,Saccostrea species such as S. glomerata, S. cucculata and S.commercialis, Mercenaria species such as M. mercenaria and M.campechensis, Ostrea species such as O. edulis, O. chilensis, and O.lurida, Arca trans versa, Panope generosa, Saxodomus nuttili, Mytilusspecies such as M. edulis (blue mussel), M. coruscus, M. chilensis, M.trossulus, and M. galloprovincialis (Mediterranean mussel), Aulacomyaater, Choromytilus chorus, Tapes semidecussatus, Perna species such asP. viridis, P. canaliculus, Venerupis species such as V. decussata, V.semidecussata, Sinonovacula constricta (Razor clam), Spisula solidissima(surf clams), Amusium balloti, Argopecten irradians (bay scallops),Pectan species such as P. alba, P. yessonsis, P. maximus, Pinctadaspecies such as P. margaritifera, P. radiate, P. maxima, P. fucata, P.albina, Hyriopsis species, such as H. cumingii, H. schlegelii,Tridacnidae species, such as Tridacna gigas (giant clam), Lamellidensspecies, such as L. marginalis (freshwater mussels), L. corrianus, andChlamys species such as C. farreri, C. opercularis, C. prpuratus and C.varia.

Although native species are preferably used, non-native species that hasalready invaded a particular body of water can also be used, e.g.,Dreissena species such as D. polymorpha (zebra mussels), D. bugenesis(quagga mussels) and D. rostiformis, Corbiucla species such as C.fluminea (Asian clam).

5.3 Methods and Systems

Provided herein are methods and systems for growing algae to extractlipids and/or produce biofuel. According to certain embodiments providedherein, the algae as described in Section 5.1 and the bivalves asdescribed in Section 5.2 are cultured for a period of time in acirculating body of water in the systems. The algae are cultured in thesame body of water as the bivalves in the system, wherein at least onedissolved inorganic or organic nutrients provided by the bivalves enablethe algae to grow more efficiently than in the absence of the bivalves.For example, the algae culture may require less carbon dioxide, nitrogenfertilizer, and/or phosphorous fertilizer. The dissolved inorganicnutrient can be carbon dioxide, ammonia, ammonium ion, a nitrite,nitrite ion, a nitrate, nitrate ion, a phosphate, or orthophosphate ion.Organic nutrients include amino acids, proteins, lipids, andcarbohydrates. However, it is not required that the bivalves and thealgae be cultured together in the same enclosure or throughout theentire culturing process. In one embodiment, the bivalves and the algaereside in the same enclosure but the bivalves reside in a zone withinthe enclosure, such as the benthic layer. In other embodiments, thebivalves and algae are cultured separately. The systems provided hereincan comprise a source of water, a plurality of enclosures and means forgrowing algae, bivalves, and/or fishes, particularly, a growth enclosurefor culturing algae, a fish enclosure for culturing planktivorousfishes, and a bivalves enclosure for culturing bivalves. The growthenclosure, the fish enclosure, and the bivalves enclosure are each influid communication with the other enclosures, and means to regulateindependently the rate, direction, or both the rate and direction, offluid flow between each of the bivalves enclosure, the growth enclosureand the fish enclosure are also provided.

The bivalves, growth, and fish enclosures can be but not limited toraceways, rectangular tanks, circular tanks, partitioned tanks, plasticbags, earthen ponds, lined ponds, channels, and artificial streams. Theenclosures of the systems provided herein are connected by channels,hoses, and pipes. The rate, direction, or both rate and direction offlow is controlled by pumps, valves, and gates. The bivalves enclosuremay comprise embedded, submerged or floating substrates, such as ropes,racks, and tubes. Preferably, the bivalves enclosure comprises asedimentary bottom and a benthic community of organisms, such that solidwastes produced by the bivalves can be remineralized by the benthicorganisms and bacteria. The enclosures provided herein can be set upaccording to knowledge known in the art, see, for example, Chapters 13and 14 in Aquaculture Engineering, Odd-Ivar Lekang, 2007, BlackwellPublishing Ltd., respectively, for description of closed productionsystems and open farming systems.

The mode of algal culture can be a batch culture, a continuous culture,or a semi-continuous culture. A batch culture comprises providing asingle inoculation of algal cells in a volume of water in the growthenclosure followed by a growing period and finally harvesting the algalpopulation when it reaches a desirable density. Typically, the growth ofalgae is characterized by a lag phase, a growth phase, and a stationaryphase. The lag phase is attributed to physiological adaptation of thealgal metabolism to growth. Cultures inoculated with exponentiallygrowing algae have short lag phases and are thus desirable. Cell densityincreases as a function of time exponentially in the growth phase. Thegrowth rate decreases as nutrient levels, carbon dioxide, unfavorablepH, or other environmental factors become limiting in a stationary phaseculture. When a growing algae culture has outgrown the maximum carryingcapacity of an enclosure, the culture can be transferred to one orseveral growth enclosures with a lower loading density. The initialalgal culture is thereby diluted allowing the algae to grow withoutbeing limited by the capacity of an enclosure. In a continuous culture,water with nutrients and gases is continuously allowed into the growthenclosure to replenish the culture, and excess water is continuouslyremoved while the algae in the water are harvested. The culture in thegrowth enclosure is maintained at a particular range of algal density orgrowth rate. In a semi-continuous culture, growing algae in an enclosureis harvested periodically followed by replenishment to about theoriginal volume of water and concentrations of nutrient and gases.Preferably, the water in the system is recycled to the bivalvesenclosure. In a preferred embodiment, the algae culture is a continuousculture, maintained in open air.

Most natural freshwater sources (rivers, lakes, springs) and municipalwater supply can be used as a source of water for used in the systemsprovided herein. Seawater, saline, and brackish water from inland,coastal or estuarine regions, irrigation water, agricultural wastewater,industrial wastewater, or municipal wastewater can also be used in thesystems provided herein. Depending on the source of water, it may benecessary to provide additional nutrients if the source is oligotrophic.In some embodiments, the source of water is eutrophic, in which case,the water comprises a plurality of algae, planktonic animals, andbacteria, and certain nutrients. Algae from the water can be used toinoculate the growth enclosure. Zooplankton that consumes algae can beremoved by filtration or by flowing the water through an enclosurecomprising zooplantivorous fishes.

The water for growing algae can comprise carbon dioxide, eitherdissolved or as bubbles, at a concentration from about 0.05% to 1% andup to 5% volume of the air introduced into the culture. The addition ofcarbon dioxide promotes photosynthesis, and helps to maintain the pH ofthe culture below pH 9. Sources of carbon dioxide include, but is notlimited to, synthetic fuel plants, gasification power plants, oilrecovery plants, ammonia plants, ethanol plants, oil refinery plants,anaerobic digestion units, cement plants, and fossil steam plants. Theuse of such carbon source in the culturing process enables carboncapture by the methods provided herein. For example, the water can besupplemented with fly ashes collected from gaseous output ofcoal-burning power generation plants. The chemical composition of flyashes depends on the source, e.g., anthracite, bituminous, and lignitecoal. Fly ashes range in size from 0.5 μm to 100 and comprise silicondioxide, calcium oxide, iron oxide, aluminum oxide. Silicon is requiredfor the growth of algae, such as the diatoms. However, according tocertain embodiments provided herein, the amount of carbon dioxiderequired to be added to the algae culture can be reduced by the presenceof a population of growing bivalves. The growth enclosures can also befertilized regularly according to conventional practices. Nutrients canbe provided in the form of fertilizers, including inorganic fertilizers,such as but not limited to, ammonium sulfate, urea, calciumsuperphosphate, and various N/P/K fertilizers (16:20:20, or 14:14:14);such as but not limited to, manure and agricultural waste. Lessfertilizer is required in the systems provided herein than a systemwithout bivalves because the bivalves excrete dissolved and solidmetabolic waste in the bivalves enclosure.

Means for culturing bivalves, algae and fishes are installed in thebivalves enclosure, the growth enclosures, and the fish enclosures, andgenerally include equipment to monitor and adjust the pH, salinity,temperature and/or dissolved gas in the water. The pH of the water ispreferably kept within the ranges of from about pH6.5 to pH9, and morepreferably from about 8.2 to about 8.7. The salinity of seawater rangespreferably from about 12 to about 40 g/L and more preferably from 20 to24 g/L. The temperature for seawater-based culture ranges preferablyfrom about 16° C. to about 27° C. or from about 18° C. to about 24° C.

According to the methods provided herein, a starter culture of algae canbe used to seed a growth enclosure. A starter culture can also be usedto inoculate a growth enclosure periodically to maintain a stablepopulation of the desired species. The starter culture is grown in waterenclosures typically smaller than the growth enclosure, referred toherein as “inoculation enclosures.” The inoculation enclosures can be,but not limited to, one or more flasks, carboys, cylinders, plasticbags, chambers, indoor tanks, outdoor tanks, indoor ponds, and outdoorponds, or a combination thereof. One or more inoculation enclosures canbe temporarily or permanently connected to one or more growth enclosuresand to each other with means for regulating fluid flow and flowdirection, e.g., gate, valve. Typically, the volume of an inoculationenclosure ranges from 1 to 10 liters, 5 to 50 liters, 25 to 150 liters,100 to 500 liters. In certain embodiments, the inoculation enclosuredoes not comprise fish.

For productive growth in an enclosure, the algae are exposed to light ofan intensity that ranges from 1000 to 10,000 lux, preferably 2500 to5000 lux. The photoperiod (light:dark in number of hours) ranges fromabout 12:12, about 14:10, about 16:8, about 18:6, about 20:4, about22:2, and up to 24:0. The light intensity and photoperiod depend on thegeographic location of the growth enclosures and the season, and may bemanipulated by artificial illumination or shading. Mixing of water inthe growth enclosure ensures that all algal cells are equally exposed tolight and nutrients. Mixing is also necessary to prevent sedimentationof the algae to the bottom or to a depth where light penetration becomeslimiting. Mixing also prevent thermal stratification of outdoorcultures. Mixing can be provided in part by the siphoning action ofbivalves or by the presence of swimming fish in the growth enclosure.Where additional mixing is required, it can be provided by any means,mechanical or otherwise, including but not limited to, paddle wheels andjet pumps.

Means and techniques for bivalves culture are well known in the art andcan be adapted to for use in certain embodiments provided herein withoutundue experimentation. For example, the bottom technique involvesscattering on the bottom of the water spat-laden cultch. Stake cultureinvolves attaching spat-laden shells to bamboo, wooden, cement, or PVCpipe stakes and driving the stakes into the bottom or laid outhorizontally. Spat may be allowed to settle directly on the stakes. Thistechnique is particularly useful in areas with soft bottoms that wouldnot allow bottom culture. Umbrella culture uses bivalves that have beenattached to ropes and suspended from a central post radiating to anchorslike spokes on a wheel, thus taking the shape of an umbrella. Rackculture is accomplished by constructing racks of treated lumber, steelrebar, or concrete blocks. Ropes, sticks, or nylon mesh bags withbivalves attached or contained are placed on the racks for growout. Themesh bag technique is used quite extensively in areas that are tooshallow for raft culture and too soft for bottom culture. Raft cultureincorporates floating structures to suspend bivalves off the bottom. Therafts can be made of logs, bamboo, Styrofoam, or 55-gallon drums. Raftmaterials are lashed together to allow flexing with wave action. Raftsare anchored to the bottom securely. Strings, ropes, nets (pearl nets,lantern nets), trays, and bags of bivalves are suspended below the raft.The long line technique also involves suspending bivalves off thebottom, wherein spat-laden cultch are attached to polypropylene rope andstrung between wooden, metal, or PVC plastic stakes inserted into thesubstrate. Other techniques known in the art can also be applied, see,for example, U.S. Pat. Nos. 3,811,411, 4,896,626, 5,511,514 and5,836,266. The bivalves can be fed with algae produced in the growthenclosure by flowing an effluent from the growth enclosure. The amountof algae allowed to flow into the bivalves enclosure is monitored andcontrolled so that the yield of the system is not affected byover-consumption by the bivalves. The amount of algae can be controlledby adjusting the flow rate of the effluent, the concentration of algaein the effluent, or the amount of time that the flow is established. Inaddition, the bivalves can also be fed with algae present in the sourceof the water, or the residual algae that are not consumed byplanktivorous fishes provided herein.

In addition to algae and bivalves, the bivalves and growth enclosure maycomprise other aquatic life, such as fishes, bacteria, zooplankton,aquatic plants, insects, worms, and nematodes. These bacteria, plants,and animals constitute various trophic levels of an ecological systemand lend stability to an algal culture grown in an open pond. However,zooplankton grazing on microalgae compete with bivalves for food and aregenerally undesirable in the growth enclosure. They can be removed fromthe water by sand filtration or controlled by keeping zooplanktivorousfishes in an enclosure. The numbers and species of planktons, includingzooplanktons, can be assessed by counting under a microscope using, forexample, a Sedgwick-Rafter cell.

In various embodiments, the algae are concentrated so that the number ofalgal cells per unit volume increases by two, five, 10, 20, 25, 30, 40,50, 75, 100-fold, or more. For example, the starting concentration of analgal culture can range from about 0.05 g/L, about 0.1 g/L, about 0.2g/L, about 0.5 g/L to about 1.0 g/L. After the concentration step, theconcentration of algae in an algal composition can range from at leastabout 0.2 g/L, about 0.5 g/L, about 1.0 g/L, about 2.0 g/L, about 5 g/Lto about 10 g/L. An alternative system to assess algal concentrationthat measures chlorophyll-a concentration (μg/L) can be used similarly.The concentration of algae can be increased progressively byconcentrating the algae in multiple stages. Starting in the growthenclosure, the algal culture is concentrated to provide an algalcomposition comprising algae at a density or concentration that ishigher than that of the algal culture in the growth enclosure. Theconcentrated algal composition can be subjected to another round ofconcentration using the same or a different technique. Although it isdesirable to remove as much water as possible from the algae beforeprocessing, it should be understood that the concentration step does notrequire that the algae be dried, dewatered, or reduced to a paste or anysemi-solid state. The resulting concentrated algae composition can be asolid, a semi-solid (e.g., paste), or a liquid (e.g., a suspension). Itcan be stored for future use, used to make biofuel, or used as feed forthe bivalves.

The choice of fish for use in the harvesting methods provided hereindepends on a number of factors, such as the palatability and nutritionalvalue of the cultured algae as food for the fishes, the lipidcomposition and content of the fish, the feed conversion ratio, the fishgrowth rate, and the environmental requirements that encourages feedingand growth of the fish. For example, it is preferable that the selectedfishes will feed on the cultured algae until satiation, and convert thealgal biomass into fish biomass rapidly and efficiently. Gut contentanalysis can reveal the dimensions of the plankton ingested by theplanktivore and the preference of the planktivore for certain species ofalgae. Knowing the average dimensions of ingested plankton, thepreference and efficiency of the planktivore towards a certain sizeclass of plankton can be determined. The size preference of aplanktivore can be used to match the dimensions of algae in the algalcomposition to improve efficiency, e.g., sizes of algae being greaterthan about 20 μm, about 20-200 μm. It may also be preferable to deploycombinations of algae and fishes that are parts of a naturally occurringtrophic system. Many trophic systems are known in the art and can beused to guide the selection of algae and fishes for use in certainembodiments provided herein.

The selected fishes should grow well in water of a salinity which issimilar to that of the algal culture, so as to reduce the need to changewater when the algae is brought to the fishes. For an open pond system,it may be preferable to use endemic species of fishes. Fishes fromdifferent taxonomic groups can be used in the growth enclosure or fishenclosure. It should be understood that, in various embodiments, fisheswithin a taxonomic group, such as a family or a genus, can be usedinterchangeably in various methods provided herein. Certain embodimentsprovided herein described below using common names of fish groups andfishes, as well as the scientific names of exemplary species. Databases,such as FishBase by Froese, R. and D. Pauly (Ed.), World Wide Webelectronic publication, www.fishbase.org, version (June 2008), provideadditional useful fish species within each of the taxonomic groups thatare useful in certain embodiments provided herein. It is contemplatedthat one of ordinary skill in art could, consistent with the scope ofcertain embodiments provided herein, use the databases to specify otherspecies within each of the described taxonomic groups for use in themethods provided herein.

In certain embodiments, the fishes used comprise fishes in the orderClupeiformes, i.e., the clupeids, which include the following families:Chirocentridae, Clupeidae (menhadens, shads, herrings, sardines, hilsa),Denticipitidae, Engraulidae (anchovies). Exemplary members within theorder Clupeiformes include but not limited to, the menhadens (Brevoortiaspecies), e.g., Ethmidium maculatum, Brevoortia aurea, Brevoortiagunteri, Brevoortia smithi, Brevoortia pectinata, Gulf menhaden(Brevoortia patronus), and Atlantic menhaden (Brevoortia tyrannus); theshads, e.g., Alosa alosa, Alosa alabamae, Alosa fallay, Alosa mediocris,Alosa sapidissima, Alos pseudoharengus, Alosa chrysochloris, Dorosomapetenense; the herrings, e.g., Etrumeus teres, Harengula thrissina,Pacific herring (Clupea pallasii pallasii), Alosa aestivalis, Ilishaafricana, Ilisha elongata, Ilisha megaloptera, Ilisha melastoma, Ilishapristigastroides, Pellona ditchela, Opisthopterus tardoore, Nematalosacome, Alosa aestivalis, Alosa chtysochloris, freshwater herring (Alosapseudoharengus), Arripis georgianus, Alosa chrysochloris, Opisthonemalibertate, Opisthonema oglinum, Atlantic herring (Clupea harengus),Baltic herring (Clupea harengus membras); the sardines, e.g., Ilishaspecies, Sardinella species, Amblygaster species, Opisthopterusequatorialis, Sardinella aurita, Pacific sardine (Sardinops sagax),Harengula clupeola, Harengula humeralis, Harengula thrissina, Harengulajaguana, Sardinella albella, Sardinella janeiro, Sardinella fimbriata,oil sardine (Sardinella longiceps), and European pilchard (Sardinapilcharthis); the hilsas, e.g., Tenuolosa species, and the anchovies,e.g., Anchoa species (A. hepsetus, A. mitchillis), Engraulis species,Thryssa species, anchoveta (Engraulis ringens), European anchovy(Engraulis encrasicolus), Engraulis eurystole, Australian anchovy(Engraulis australis), and Setipinna phasa, Coilia dussumieri.

In a preferred embodiment, the fishes used are shiners. A variety ofshiners that inhabit the Gulf of Mexico, particularly Northern Gulf ofMexico, can be used. Examples of shiners include but are not limited to,members of Luxilus, Cyprinella and Notropis genus, Alabama shiner(Cyprinella callistia), Altamaha shiner (Cyprinella xaenura), Amecashiner (Notropis amecae), Ameca shiner (Notropis amecae), Apalacheeshiner (Pteronotropis grandipinnis), Arkansas River shiner (Notropisgirardi), Aztec shiner (Aztecula sallaei old), Balsas shiner (Hybopsisboucardi), Bandfin shiner (Luxilus zonistius), Bannerfin shiner(Cyprinella leedsi), Beautiful shiner (Cyprinella formosa), Bedrockshiner (Notropis rupestris), Bigeye shiner (Notropis hoops), Bigmouthshiner (Hybopsis dorsalis), Blackchin shiner (Notropis heterodon),Blackmouth Shiner (Notropis melanostomus), Blacknose shiner (Can QuebecNotropis heterolepis), Blacknose shiner (Notropis heterolepis),Blackspot shiner (Notropis atrocaudalis), Blacktail shiner (Cyprinellavenusta), Blacktip shiner (Lythrurus atrapiculus), Bleeding shiner(Luxilus zonatus), Blue Shiner (Cyprinella caerulea), Bluehead Shiner(Pteronotropis hubbsi), Bluenose Shiner (Pteronotropis welaka),Bluestripe Shiner (Cyprinella callitaenia), Bluntface shiner (Cyprinellacamura), Bluntnose shiner (Notropis simus), Bluntnosed shiner (Selenesetapinnis), Bridle shiner (Notropis bifrenatus), Broadstripe shiner(Notropis euryzonus), Burrhead shiner (Notropis asperifrons), CahabaShiner (Notropis cahabae), Cape Fear Shiner (Notropis mekistocholas),Cardinal shiner (Luxilus cardinalis), Carmine shiner (Notropispercobromus), Channel shiner (Notropis wickliffi), Cherryfin shiner(Lythrurus roseipinnis), Chihuahua shiner (Notropis chihuahua), Chubshiner (Notropis potter)), Coastal shiner (Notropis petersoni),Colorless Shiner (Notropis perpallidus), Comely shiner (Notropisamoenus), Common emerald shiner (Notropis atherinoides), Common shiner(Luxilus cornutus), Conchos shiner (Cyprinella panarcys), Coosa shiner(Notropis xaenocephalus), Crescent shiner (Luxilus cerasinus), CuatroCienegas shiner (Cyprinella xanthicara), Durango shiner (Notropisaulidion), Dusky shiner (Notropis cummingsae), Duskystripe shiner(Luxilus pilsbryi), Edwards Plateau shiner (Cyprinella lepida), Emeraldshiner (Notropis atherinoides), Fieryblack shiner (Cyprinellapyrrhomelas), Flagfin shiner (Notropis signipinnis), Fluvial shiner(Notropis edwardraneyi), Ghost shiner (Notropis buchanani), Gibbousshiner (Cyprinella garmani), Golden shiner (Notemigonus crysoleucas),Golden shiner minnow (Notemigonus crysoleucas), Greenfin shiner(Cyprinella chloristia), Greenhead shiner (Notropis chlorocephalus),Highfin shiner (Notropis altipinnis), Highland shiner (Notropismicropteryx), Highscale shiner (Notropis hypsilepis), Ironcolor shiner(Notropis chalybaeus), Kiamichi shiner (Notropis ortenburgeri), Lakeemerald shiner (Notropis atherinoides), Lake shiner (Notropisatherinoides), Largemouth shiner (Cyprinella bocagrande), Longnoseshiner (Notropis longirostris), Mexican red shiner (Cyprinella rutila),Mimic shiner (Notropis volucellus), Mirror shiner (Notropisspectrunculus), Mountain shiner (Lythrurus lirus), Nazas shiner(Notropis nazas), New River shiner (Notropis scabriceps), Ocmulgeeshiner (Cyprinella callisema), Orangefin shiner (Notropis ammophilus),Orangetail shiner (Pteronotropis merlini), Ornate shiner (Cyprinellaornata), Ouachita Mountain Shiner (Lythrurus snelsoni), Ouachita shiner(Lythrurus snelsoni), Ozark shiner (Notropis ozarcanus), Paleband shiner(Notropis albizonatus), Pallid shiner (Hybopsis amnis), Peppered shiner(Notropis perpallidus), Phantom shiner (Notropis orca), Pinewoods shiner(Lythrurus matutinus), Plateau shiner (Cyprinella lepida), Popeye shiner(Notropis ariommus), Pretty shiner (Lythrurus bellus), Proserpine shiner(Cyprinella proserpina), Pugnose shiner (Notropis anogenus), Pygmyshiner (Notropis tropicus), Rainbow shiner (Notropis chrosomus), RedRiver shiner (Notropis bairdi), Red shiner (Cyprinella lutrensis),Redfin shiner (Lythrurus umbratilis), Redlip shiner (Notropischiliticus), Redsi de shiner (Richardsonius balteatus), Ribbon shiner(Lythrurus fumeus), Rio Grande bluntnose shiner (Notropis simus), RioGrande shiner (Notropis jemezanus), River shiner (Notropis blennius),Rocky shiner (Notropis suttkusi), Rosefin shiner (Lythrurus ardens),Rosyface shiner (Notropis rubellus), Rough shiner (Notropis baileyi),Roughhead Shiner (Notropis semperasper), Sabine shiner (Notropissabinae), Saffron shiner (Notropis rubricroceus), Sailfin shiner(Notropis hypselopterus), Salado shiner (Notropis saladonis), Sandshiner (Notropis stramineus), Sandbar shiner (Notropis scepticus),Satinfin shiner (Cyprinella analostana), Scarlet shiner (Lythrurusfasciolaris), Sharpnose Shiner (Notropis oxyrhynchus), Notropisatherinoides, Notropis hudsonius, Richardsonius balteatus, Pomoxisnigromaculatus, Cymatogaster aggregata, Shiner Mauritania (Selenedorsalis), Silver shiner (Notropis photogenis), Silver shiner(Richardsonius balteatus), Silver shiner (Richardsonius balteatus),Silver shiner (Notropis photogenis), Silverband shiner (Notropisshumardi), Silverside shiner (Notropis candidus), Silverstripe shiner(Notropis stilbius), Skygazer shiner (Notropis uranoscopus), SmalleyeShiner (Notropis buccula), Soto la Marina shiner (Notropisaguirrepequenoi), Spotfin shiner (Cyprinella spiloptera), Spottailshiner (Notropis hudsonius), Steelcolor shiner (Cyprinella whipplei),Striped shiner (Luxilus chrysocephalus), Swallowtail shiner (Notropisprocne), Taillight shiner (Notropis maculatus), Tallapoosa shiner(Cyprinella gibbsi), Tamaulipas shiner (Notropis braytoni), Telescopeshiner (Notropis telescopus), Tennessee shiner (Notropis leuciodus),Tepehuan shiner (Cyprinella alvarezdelvillari), Texas shiner (Notropisamabilis), Topeka shiner (Notropis topeka), Tricolor shiner (Cyprinellatrichroistia), Turquoise Shiner (Erimonax monachus), Warpaint shiner(Luxilus coccogenis), Warrior shiner (Lythrurus alegnotus), Wedgespotshiner (Notropis greenei), Weed shiner (Notropis texanus), White shiner(Luxilus albeolus), Whitefin shiner (Cyprinella nivea), Whitemouthshiner (Notropis alborus), Whitetail shiner (Cyprinella galactura),Yazoo shiner (Notropis rafinesquei), Yellow shiner (Cymatogasteraggregata), Yellow shiner (Notropis calientis), and Yellowfin shiner(Notropis lutipinnis).

Other exemplary fish species that can be used to harvest algae include:Brevoortia species such as B. patronus and B. tyrannus, Hyporhamphusunifasciatus, Sardinella aurita, Adinia xenica, Diplodus holbrooki,Dorosoma petenense, Lagodon rhombodides, Microgobius gulosus, Mugilspecies such as Mugil cephalus, Mugil cephalus, Mugil curema,Sphoeroides species such as Sphoeroides maculatus, Sphoeroides nephelus,Sphoeroides parvus, Sphoeroides spengleri, Aluterus schoepfi, Anguillarostrata, Arius felis, Bairdella chrysoura, Bairdeiella chrysoura,Chasmodies species such as Chasmodes saburrae and Chasmodies saburrae,Diplodus holbrooki, Heterandria formosa, Ilybopsis winchelli, Ictalurusspecies such as Ictalurus serracantus and Ictalurus punctatus,Leiostomus xanthurus, Micropogonias undulatus, Monacanthus ciliatus,Notropis texanus, Opisthonema oglinum, Orthopristis chrysoptera,Stephanolepis hispidus, Syndous foetens, Syngnathus species such asSyngnathus scovelli, Trinectes maculatus, Archosargus probatocephalus,Carpiodes species such as C. cyprinus and C. velifer, Dorosomacepedianum, Erimyzon species such as Erimyzon oblongus, Erimyzonsucetta, and Erimyzon tenuis, Floridichthys carpio, Microgobius guloses,Monacanthus cilatus, Moxostoma poecilurum, and Orthopristischrysophtera.

Transgenic fish and genetically improved fish can also be used in theculturing and/or harvesting methods provided herein. The term“genetically improved fish” refers herein to a fish that is geneticallypredisposed to having a higher growth rate and/or a lipid content thatis higher than a wild type fish, when they are cultured under the sameconditions. Such fishes can be obtained by traditional breedingtechniques or by transgenic technology. Over-expression or ectopicexpression of a piscine growth hormone transgene in a variety of fishesresulted in enhanced growth rate. For example, the growth hormone genesof Chinook salmon, Sockeye salmon, Tilapia, Atlantic salmon, grass carp,and mud loach have been used in creating transgenic fishes (Zbikowska,Transgenic Research, 12:379-389, 2003; Guan et al., Aquaculture,284:217-223, 2008). Transgenic carp or transgenic tilapia comprising anectopically-expressed piscine growth hormone transgene are particularlyuseful in the harvesting methods provided herein.

Examples of means and methods for processing lipids such as algal oiland fish lipids into biofuel, such as biodiesel, can be found in thefollowing patent publications, the entire contents of each of which areincorporated by reference herein: U.S. Patent Publication No.2007/0010682, entitled “Process for the Manufacture of Diesel RangeHydrocarbons;” U.S. Patent Publication No. 2007/0131579, entitled“Process for Producing a Saturated Hydrocarbon Component;” U.S. PatentPublication No. 2007/0135316, entitled “Process for Producing aSaturated Hydrocarbon Component;” U.S. Patent Publication No.2007/0135663, entitled “Base Oil;” U.S. Patent Publication No.2007/0135666, entitled “Process for Producing a Branched HydrocarbonComponent;” U.S. Patent Publication No. 2007/0135669, entitled “Processfor Producing a Hydrocarbon Component;” and U.S. Patent Publication No.2007/0299291, entitled “Process for the Manufacture of Base Oil.”

In certain embodiments, the extraction of lipids from the fishes cancomprise heating the fish to a temperature between 70° C. to 100° C.,pressing the fishes to release the lipids, and collecting the lipids.Separation of the lipids from an aqueous phase and/or a solid phase canbe included in the extraction step. The entire fish or a portion thereofcan be used to extract lipids. If lipid concentrates are desired,several established methods could be employed, including chromatography,fractional or molecular distillation, enzymatic splitting,low-temperature crystallization, supercritical fluid extraction, or ureacomplexation.

In certain embodiments, the processing step involves heating the fishesto greater than about 70° C., 80° C., 90° C. or 100° C., typically by asteam cooker, which coagulates the protein, ruptures the fat depositsand liberates lipids and oil and physico-chemically bound water, and;grinding, pureeing and/or pressing the fish by a continuous press withrotating helical screws. The fishes can be subjected to gentle pressurecooking and pressing which use significantly less energy than that isrequired to obtain lipids from algae. The coagulate may alternatively becentrifuged. This step removes a large fraction of the liquids (pressliquor) from the mass, which comprises an oily phase and an aqueousfraction (stickwater). The separation of press liquor can be carried outby centrifugation after the liquor has been heated to 90° C. to 95° C.Separation of stickwater from oil can be carried out in vertical disccentrifuges. The lipids in the oily phase (fish oil) may be polished bytreating with hot water which extracts impurities from the lipids.

In certain embodiments, the extracted fish lipids are not limited to useas biofuels. In one embodiment, the extracted fish lipids can be used toobtain omega 3 fatty acids, such as eicosahexaenoic acid (EPA) and/ordocosahexaenoic acid (DHA) and/or derivatives thereof including, but notlimited to esters, glycerides, phospholipids, sterols, and/or mixturesthereof. In one embodiment, the extracted fish lipids contain EPA and/orDHA ranging from 1 to 50%, depending on the fish species, age, location,and a host of ecological and environmental factors. If higher EPA and/orDHA concentrations are desired, several established methods could beemployed, including chromatography, fractional or moleculardistillation, enzymatic splitting, low-temperature crystallization,supercritical fluid extraction, or urea complexation. These methods canfurther concentrate the EPA and/or DHA to nearly pure EPA and/or DHA.

In certain embodiments, EPA- and/or DHA-containing lipids may beseparated and concentrated by short-path distillation, or moleculardistillation. The lipids are first transesterified, either acid- orbase-catalyzed, with ethanol to produce a mixture of fatty acid ethylesters (FAEE). The FAEE are then fractionated in the short-pathdistillation to remove the short chain FAEE, C-14 to C-18. Theconcentrate of FAEE from C-20 to C-22 is where the EPA and/or DHA can befound. A second distillation of the concentrate can result in a finalOmega-3 content of up to 70%. The concentration of the EPA and/or DHA inthe final product will depend on the initial lipid profile of the fishoil. The FAEE can be used as a consumer product at this stage (fish oilcapsules). In some countries, the FAEE are required to be reconverted totriglycerides through a glycerolysis reaction before they can be sold asa consumer product. In order to obtain pure EPA and/or DHA, anadditional purification step is required using chromatography, enzymatictransesterification, ammonia complexation, or supercritical fluidextraction.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

1-29. (canceled)
 30. A method for extracting lipids from planktivorousfishes comprising: a. deploying planktivorous fishes into a body ofwater comprising algae and bivalves; b. harvesting the algae by feedingthe algae to the planktivorous fishes; c. harvesting the planktivorousfishes; and d. extracting lipids from the planktivorous fishes.
 31. Themethod of claim 30, wherein the extracting comprises heating theplanktivorous fishes to a temperature of between 70° C. and 100° C.,pressing the fishes to release the lipids, and collecting the lipids.32. The method of claim 30, further comprising processing the lipids bya technique selected from the group consisting of chromatography,fractional distillation, molecular distillation, enzymatic splitting,low-temperature crystallization, supercritical fluid extraction, andurea complexation.
 33. The method of claim 30, further comprisingpolishing the lipids to form a biofuel.
 34. The method of claim 33,wherein the biofuel is biodiesel.
 35. The method of claim 30, furthercomprising processing the lipids to form a product comprising at leastone of EPA and DHA.
 36. The method of claim 35, wherein the processingcomprises transesterifying the lipids.
 37. The method of claim 35,wherein the processing comprises removing C₁₄ to C₁₈ fatty acid ethylesters from the lipids.
 38. The method of claim 35, wherein theprocessing comprises at least two distillation steps.
 39. The method ofclaim 35, wherein the product is for human consumption or animal feed.40. The method of claim 30, wherein the planktivorous fishes or thebivalves are used as food.
 41. The method of claim 30, wherein the bodyof water is a eutrophic zone.
 42. The method of claim 30, wherein thebody of water is an open farming system.
 43. The method of claim 30,wherein the planktivorous fishes are from a local aquatic trophicsystem.
 44. The method of claim 30, wherein the planktivorous fishes andthe algae are parts of a naturally occurring trophic system.
 45. Themethod of claim 30, wherein the planktivorous fishes are selected fromthe group consisting of Brevoortia patronus, Clupea harengus, andcombinations thereof.
 46. The method of claim 30, wherein theplanktivorous fishes are transgenic
 47. The method of claim 30, whereinthe algae composition in the body of water changes seasonally.
 48. Themethod of claim 30, wherein the algae are of a size selected from thegroup consisting of greater than about 20 μm and about 20 to 200 μm. 49.The method of claim 30, wherein the algae are fed to the bivalves at arate such that the algae produced in the system is greater than when thealgae is cultured in the system without the bivalves.
 50. The method ofclaim 30, wherein the bivalves filter algae of a size smaller than thealgae filtered by the planktivorous fishes.
 51. The method of claim 30,wherein the bivalves are selected from the group consisting ofpseudofeces-producing bivalves, filter-feeding bivalves, deposit-feedingbivalves, and combinations thereof.
 52. The method of claim 30, whereinthe bivalves are selected from the group consisting of Mytilus edulis,Crassostrea virginica, and combinations thereof.
 53. The method of claim30, wherein the salinity of the body of water is selected from the groupconsisting of about 12 to about 40 g/L and about 20 to about 24 g/L. 54.The method of claim 30, wherein the temperature of the body of water isselected from the group consisting of about 16° C. to about 27° C. andabout 18° C. to about 24° C.
 55. The method of claim 30, wherein theplanktivorous fishes are selected based on a factor selected from thegroup consisting of the lipid composition and content of the fishes, thefeed conversion ratio, the growth rate of the fishes, and combinationsthereof.